Description of Research Expertise

A mouse model of human L1 retrotransposition as a tool for insertional mutagenesis and discovery of gene function.

The SVA element is a non-autonomous retrotransposon that can cause disease

Preclinical trials of AAV-mediated gene therapy of hemophilia A in mice and dogs.

Key words: hemophilia, retrotransposon, gene therapy.

Description of Research

Dr. Kazazian has had a long interest in the nature of retrotransposable elements in humans. These L1 elements are present in 500,000 inactive copies and 80-100 active copies in the average human genome. The active elements encode a reverse transcriptase, an endonuclease, and other protein functions that help them retrotranspose. Retrotransposition is characterized by transcription of the L1 DNA into L1 RNA, reverse transcription to L1 cDNA, and integration into a new site in the genome. Some retrotranspositions produce disease, such as hemophilia A, muscular dystrophy, and breast and colon cancer. The lab has developed an assay for retrotransposition in human and rodent cells in culture and has used this assay to elucidate important L1 protein sequences for retrotransposition and the number of human L1 elements capable of retrotransposition. It has also used the assay to demonstrate the existence of 3 distinct subfamilies of mouse L1 elements and shown that mice have up to 3,000 active L1s or 40 times the number present in the human genome. We have also shown that L1 retrotransposition is a likely source of exon shuffling via transduction of sequences 3´ to active L1s into new genomic sites. This shuffling of DNA sequences from one place to another suggests a major evolutionary benefit of retrotransposons to their mammalian hosts. Recently, we have successfully created a mouse model of human L1 retrotransposition. These mice demonstrate retrotransposition of marked L1s in male germ cells at frequencies as high as 1 in 20 sperm. In addition, the events are indistinguishable from natural endogenous insertions. We plan to use this model of insertional mutagenesis to characterize phenotypic effects of various mouse genes.

A most exiting recent project uses computational and wet bench skills along with high-throughput sequencing. In this work we find essentially all of the L1 insertion sites in any genome using PCR reactions off the 3' end of L1 and Solexa sequencing of the products. With this approach we have already found 600 L1s that are polymorphic as to presence in human genomes. We are using the approach to discover the frequency of new insertions in the population, the frequency of somatic insertions, the frequency of new insertions in clonal stem cell lines,and the frequency of insertions in cancers, other diseases, and aborted fetuses. We have obtained a Challenge Grant to augment Genome Wide Association studies with L1 and Alu insertional polymorphisms.

The lab has long studied the molecular diagnosis and treatment of hemophilia A. The lab has made a knockout mouse model of hemophilia A for studies of factor VIII biology and gene therapy. We are now working on treatment of the mice by factor VIII derived from skin and liver. We have shown that factor VIII transgenes expressed in the outer layers of skin will correct factor VIII deficiency in knockout mice. We have also obtained correction of factor VIII deficiency in the mice using adenoviral vectors and transient immune suppression. Presently, we are using gene therapy with adeno-associated virus vectors to correct hemophilia A in these mice and hemophilia A dogs, and have obtained 100% correction of the mice over 1 year.